The identification and sequencing of polypeptides has become of increased importance with the rapid development of the field of proteomics, wherein the expression products of novel genes are examined as to their function and composition.
Matrix-assisted laser desorption ionization (MALDI) mass spectrometry is a method developed for peptide and polypeptide sequencing. (For a reference to the principles of MALDI mass spectrometry, see e.g. Spengler et al., “Peptide Sequencing by Matrix-assisted Laser-desorption Mass Spectrometry”, Rapid Communications in Mass Spectrometry, Vol. 6, pp. 105–108 (1992).) MALDI mass spectrometry offers several advantages in the field of mass spectrometry. For example, it provides a higher sensitivity than the conventional electrospray triple quadropole equipment. When used in combination with time-of-flight (TOF) mass analyzers, MALDI mass spectrometry is also applicable to higher mass peptides than can be analyzed with triple quadropole equipment. MALDI mass spectrometry is also useful for analyzing complex mixtures with minimal sample purification. Electrospray ionization, on the other hand, is readily interfaced to powerful separation techniques including liquid chromatography (LC) and various forms of capillary electrophoresis (CE). Highly automated analyses are possible when using LC and CE as the sample purification and introduction devices.
However, current MALDI and, to a lesser extent, electrospray ionization mass spectro-metric methods fail to adequately offer predictable tandem mass spectrometry fragmentation patterns. For example, multiple ion series (including a-ions, b-ions, and y-ions) are often observed, resulting in MALDI post-source decay spectra that are too complex for efficient interpretation and sequencing. Multiple ion series (b- and y-ions), plus internal fragments and both singly and multiply charged ions are formed from multiply charged precursor ions generated by electrospray ionization, and the resulting tandem mass spectra are often difficult to interpret de novo. Accordingly, problems with fragmentation have limited the ability to rapidly sequence polypeptides using mass spectrometry. As a result, mass spectrometry, and particularly MALDI mass spectrometry, has been of limited value in this area.
Several research groups have attempted to improve the utility of mass spectrometry in the field of polypeptide sequencing through the use of chemical derivatization techniques. Such techniques have been utilised to promote and direct fragmentation in the MSMS spectra of peptides with the goal of increasing sensitivity and decreasing the complexity of the resulting spectra. Most of these methods provide cationic derivatives. For example, derivatization with a quaternary ammonium group, and analysis using the static SIMS ionization method has been suggested. However, application of such techniques using MALDI mass spectrometry and electrospray ionization with low-energy collisional activation have not proven generally effective.
More recently, for the determination of an amino acid sequence, Keough et al (WO 00/43792, in the name of The Procter & Gamble Company) have suggested a derivatization of the N-terminus of a polypeptide with one or more acidic moieties having pKa values of less than 2 before analysis by mass spectrometry of the analyte, such as with MALDI mass spectrometry. The acidic moiety is preferably a sulfonic acid or a disulfonic acid derivative. The derivatives promote a charge-site initiated cleavage of backbone amide bonds and they enable the selective detection of only a single series of fragment ions comprising the y-ions. However, the reaction according to Keough et al are generally performed under non-aqueous conditions due to the poor water stability of the reagents utilized therein. Accordingly, for a commercially useful determination of amino acid sequences by mass spectrometry, there is still a need for improved methods that fulfill the requirements especially for automated procedures.